1. Field of the Invention
The present invention relates to a biosensor and a method of quantitative analysis of a material which relates to a reaction of a specific compound in a liquid sample a biologically active substance such as an enzyme.
2. Description of the Related Art
When a biological liquid sample such as blood is analyzed using a biosensor which comprises electrochemical detecting means, a reducing compound such as ascorbic acid or uric acid which is present in the sample has an electrochemical or chemical interference on the analysis, which is always a problem in the analysis.
Hitherto, various measures to remove or suppress such interference have been proposed in patent specifications and literatures. They are summarized as follows:
(1) Use of an interference-removing membrane: PA1 (2) Electrode oxidation: PA1 (3) Use of plural working electrodes: PA1 (4) Addition of an enzyme for oxidizing an interfering substance: PA1 (5) Double potential step method: PA1 (1) Use of an interference-removing membrane: PA1 (2) Electrode oxidation: PA1 (3) Use of plural working electrodes: PA1 (4) Addition of an enzyme for oxidizing an interfering substance: PA1 (5) Double potential step method:
U.S. Pat. Nos. 3,979,274 and 4,240,889, Japanese Patent Kokai Publication Nos. 211542/1982 and 5643/1983, etc.
U.S. Pat. No. 4,431,507, Japanese Patent Kokai Publication Nos. 118152/1982, 211054/1982, 5642/1983, 85148/1983, 85149/1983 and 146847/1983, The 11th Chemical Sensor Symposium, Okawa et al, 24. "Electrochemical On-Line Elimination of Electroactive Interference for Flow-Type Biosensor System", etc.
U.S. Pat. No. 3,539,455, Japanese Patent Kokai Publication Nos. 146847/1983 and 253648/1989, Miyahara et al, Sensor and Actuators, 7, 1 (1985), etc.
Japanese Patent Publication No. 17427/1983
The 58th Spring Annual Meeting of the Japan Chemical Society, 4IG06, Matsuura et al, "Measurement of Hydrogen Peroxide with A Micro Carbon Fiber Electrode".
However, each of the above measures has its own drawbacks as follows:
In this method, an electrode which is an electrochemically detecting device is covered with a selectively permeable membrane, whereby a substance to be analyzed permeates the membrane while concomitant interfering substances do not. This method can be employed when a substance having a very low molecular weight such as oxygen molecules or hydrogen peroxide is used as an electrochemically reactive substance. But, when a mediator for electric charges such as potassium ferricyanide or ferrocene is used, this method cannot be applied since the concomitant interfering substance and the mediator cannot be distinguished according to their sizes. Further, this method cannot be a remedy for an oxidation-reduction reaction between the concomitant substance and the mediator which takes place outside the interference-removing membrane, namely in the sample liquid. In addition, the membrane may decrease a sensitivity and a response of the electrode and a degree of such deterioration depends on a thickness of the membrane so that a difference between individual sensors is enlarged.
This method requires an additional electrode system for anodizing the concomitant interfering substance in the sample (an electrolytic electrode system) in addition to an electrode system for measuring an object substance (a measuring electrode system). When the sample is supplied to a measuring system, the interfering substance is anodized by the electrolytic electrode system before it reaches an enzyme reaction system or the measuring electrode system.
Since this method essentially requires the electrolytic electrode system in addition to the measuring electrode system, and two electrode systems and the reaction system of the biologically active substance such as an enzyme are spacially separated, the sensor has a complicated structure. To increase an electrolytic efficiency of the interfering substance, a surface area of the electrolytic electrode is increased, or the sample liquid is intentionally stirred or flowed. However, the structure of the sensor is complicated and enlarged, or the response is decreased. This method may not be suitable for a disposable sensor.
The increase of the electrolytic efficiency of the interfering substance is contrary to the reduction of the measuring time and the increase of the response. To satisfy both requirements, a very thin integrated porous electrode system is proposed. But, since such thin electrode is weak and unstable, it requires reinforcement of the structure so that it is difficult to supply a simple and cheap sensor.
Since the sensor as a whole has the electrolytic electrode system in addition to the measuring electrode system, electric circuits and a measuring software become complicated and expensive.
In this method, an electrode system for measuring the interfering substance present in the sample is used in addition to the measuring electrode system. When the sample is supplied, the measuring electrode system measures signals from both the object substance and the interfering substance while the electrode system for measuring the interfering substance measures only the signal from the interfering substance. Then, a difference between these two measured value is calculated to give a concentration of the object substance to be measured.
This method essentially requires the electrode system for measuring the interfering substance. Since there is a possibility that a reaction product or reaction products produced by the measuring electrode system may have some influence on the electrode system for measuring the interfering substance, these two electrode systems should be spacially separated with a sufficient distance. This results in enlargement and a mote complicated structure of the whole sensor. Since two or more electrode systems are used, two or more electric circuits for amplifying detected currents are necessary.
Further, measuring sensitivities for the object substance measurement and the interfering substance measurement should be matched, but such matching of the sensitivities is very difficult practically. In the case of a repetitive use sensor, the sensitivities of the electrode systems for measuring the interfering substance may be calibrated, but such calibration is impossible for the disposable sensor.
In this method, the interfering substance such as ascorbic acid or uric acid is oxidized with a respective oxidase before it participates in the electrode reaction or the oxidation-reduction reaction with the substance to be measured. Since a highly specific enzyme is used to remove the interfering substance in this method, plural enzymes should be used when plural interfering substances are present in the sample. This leads to the increase of a production cost of the biosensor. The preoxidation of the interfering substance is essential, and it is necessary to prevent interference of the measurement of the object substance caused by a product from oxidation of the interfering substance. Therefore, the sensor has a complicated structure inevitably. In addition, the interfering substance is removed through a conversion by the oxidation to a material which cannot be measured. This means that some information, which may be valuable if measured, is discarded.
When a natural potential (E.sub.02) of the measuring electrode against the object substance to be measured and a natural potential (E.sub.01) against the interfering substance are different (assuming E.sub.01 &lt;E.sub.02), the concentration of the interfering substance is measured at a potential E.sub.1 which satisfies E.sub.01 &lt;E.sub.1 &lt;E.sub.02, while a total concentration of the object substance and the interfering substance is measured at a potential E.sub.2 which is larger than E.sub.02 (E.sub.02 &lt;E.sub.2), and then a difference between E.sub.1 and E.sub.2 is calculated to obtain the concentration of the object substance.
According to the measuring principle of this method, if the natural potential E.sub.02 against the object substance and the natural potential E.sub.01 against the interfering substance are not sufficiently different, the concentration of the object substance and the total concentration of the object substance and the interfering substance cannot be separated and measured. When the object substance to be measured is hydrogen peroxide, the above potential relationship can be often established. Depending on an electrode substance or a surface condition of the electrode, E.sub.01 and E.sub.02 are very close to each other or sometimes E.sub.01 exceeds E.sub.02. To achieve stability or expansion of a linear range of the biosensor, the mediator is often used. In such case, the electric charges are transferred with the mediator between the electrode and the object substance to be measured or the interfering substance, E.sub.01 and E.sub.02 are equal. Therefore, the double potential step method cannot be used.